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1.
BMC Infect Dis ; 20(1): 587, 2020 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-32770954

RESUMO

BACKGROUND: Tuberculosis (TB) is transmitted in bioaerosols containing Mycobacterium tuberculosis (Mtb). Despite being central to ongoing TB transmission, no routine diagnostic assay exists to measure Mtb in bioaerosols. Furthermore, published studies of Mtb in bioaerosol samples have been limited to individuals with sputum-positive pulmonary TB. Notably, TB diagnosis is based on clinical symptoms and sputum laboratory findings. This is despite the fact that approximately half of all patients commencing TB treatment are sputum-negative, resulting in a high proportion of presumptive treatments. Here, we propose to use a sensitive air sampling protocol to investigate the prevalence of Mtb-containing bioaerosols in both sputum-positive and sputum-negative TB suspects, at the same time evaluating the potential to identify unrecognized transmitters of TB. METHODS: Our parallel-group design will identify viable Mtb in bioaerosols produced by individuals attending a TB clinic in South Africa. Sampling will be performed on eligible individuals presenting with symptoms indicative of TB and repeated at 14 days if initially positive. Participants will be prospectively classified into three distinct groups based on National TB Control Program (NTBCP) criteria: Group A, TB notification with sputum-based laboratory confirmation; Group B, TB notification with empiric diagnosis; and Group C, individuals not notified. Group C individuals with detectable Mtb bioaerosol will be monitored until resolution of clinical and laboratory status. Collection of bioaerosol specimens will be via two consecutive sampling modalities: (1) direct sampling following a specific respiratory manoeuvre; and (2) indirect sampling during passive respiratory activity. Bioaerosol specimens will be analyzed for viable Mtb using DMN-trehalose staining and live-cell fluorescence microscopy. Mtb genomes and mycobacterial and host lipids will be detected using droplet digital PCR and mass spectrometry analyses, respectively. The primary objective is to determine the prevalence of Mtb bioaerosols in all TB clinic attendees and in each of the groups. Secondary objectives are to investigate differences in prevalence of Mtb bioaerosol by HIV status and current isoniazid preventive therapy (IPT) use; we will also determine the impact of anti-TB chemotherapy on Mtb-containing bioaerosol production. DISCUSSION: Respiratory bioaerosol has a potential role in non-invasive TB diagnosis, infectivity measurement and treatment monitoring. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04241809 . Date of Registration: 27/1/2020.


Assuntos
Aerossóis/análise , Manejo de Espécimes/métodos , Tuberculose Pulmonar/diagnóstico , Adulto , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase , África do Sul , Escarro/microbiologia
2.
Mol Cell ; 79(3): 416-424.e5, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645367

RESUMO

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Genoma Bacteriano/imunologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/classificação , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
3.
Nat Commun ; 11(1): 3703, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32710080

RESUMO

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control.


Assuntos
Acil Coenzima A/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/química , Acil Coenzima A/metabolismo , Sítio Alostérico , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cristalografia por Raios X , DNA Bacteriano/química , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/metabolismo , Ligantes , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/metabolismo
4.
Nat Commun ; 11(1): 3576, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681021

RESUMO

CRISPR/Cas9 is a programmable genome editing tool widely used for biological applications and engineered Cas9s have increased discrimination against off-target cleavage compared with wild-type Streptococcus pyogenes (SpCas9) in vivo. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. We show that DNA cleavage is impaired by more than 100- fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the observed rate of cleavage without increasing the rate of DNA rewinding and release. The kinetic partitioning favors release rather than cleavage of a bound off-target substrate only because the cleavage rate is so low. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , DNA Bacteriano/metabolismo , Streptococcus pyogenes/enzimologia , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Clivagem do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Cinética , Streptococcus pyogenes/química , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(25): 14202-14208, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513722

RESUMO

FtsK protein contains a fast DNA motor that is involved in bacterial chromosome dimer resolution. During cell division, FtsK translocates double-stranded DNA until both dif recombination sites are placed at mid cell for subsequent dimer resolution. Here, we solved the 3.6-Å resolution electron cryo-microscopy structure of the motor domain of FtsK while translocating on its DNA substrate. Each subunit of the homo-hexameric ring adopts a unique conformation and one of three nucleotide states. Two DNA-binding loops within four subunits form a pair of spiral staircases within the ring, interacting with the two DNA strands. This suggests that simultaneous conformational changes in all ATPase domains at each catalytic step generate movement through a mechanism related to filament treadmilling. While the ring is only rotating around the DNA slowly, it is instead the conformational states that rotate around the ring as the DNA substrate is pushed through.


Assuntos
DNA Bacteriano/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Translocação Genética/fisiologia , Divisão Celular/fisiologia , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Microscopia Crioeletrônica , DNA/química , DNA Bacteriano/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Conformação Proteica
6.
Proc Natl Acad Sci U S A ; 117(25): 14322-14330, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32518115

RESUMO

Phosphorothioate (PT) DNA modifications-in which a nonbonding phosphate oxygen is replaced with sulfur-represent a widespread, horizontally transferred epigenetic system in prokaryotes and have a highly unusual property of occupying only a small fraction of available consensus sequences in a genome. Using Salmonella enterica as a model, we asked a question of fundamental importance: How do the PT-modifying DndA-E proteins select their GPSAAC/GPSTTC targets? Here, we applied innovative analytical, sequencing, and computational tools to discover a novel behavior for DNA-binding proteins: The Dnd proteins are "parked" at the G6mATC Dam methyltransferase consensus sequence instead of the expected GAAC/GTTC motif, with removal of the 6mA permitting extensive PT modification of GATC sites. This shift in modification sites further revealed a surprising constancy in the density of PT modifications across the genome. Computational analysis showed that GAAC, GTTC, and GATC share common features of DNA shape, which suggests that PT epigenetics are regulated in a density-dependent manner partly by DNA shape-driven target selection in the genome.


Assuntos
Bactérias/genética , Bactérias/metabolismo , DNA Bacteriano/metabolismo , Epigênese Genética/fisiologia , Epigenômica , Fosfatos/metabolismo , 2-Aminopurina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Consenso , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Genoma Bacteriano , Salmonella enterica/genética
7.
J Med Microbiol ; 69(8): 1105-1113, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32597748

RESUMO

Introduction. Burkholderia cepacia complex (Bcc) bacteria, currently consisting of 23 closely related species, and Burkholderia gladioli, can cause serious and difficult-to-treat infections in people with cystic fibrosis. Identifying Burkholderia bacteria to the species level is considered important for understanding epidemiology and infection control, and predicting clinical outcomes. Matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF) is a rapid method recently introduced in clinical laboratories for bacterial species-level identification. However, reports on the ability of MALDI-TOF to accurately identify Bcc to the species level are mixed.Aim. The aim of this project was to evaluate the accuracy of MALDI-TOF using the Biotyper and VITEK MS systems in identifying isolates from 22 different Bcc species and B. gladioli compared to recA gene sequencing, which is considered the current gold standard for Bcc.Methodology. To capture maximum intra-species variation, phylogenetic trees were constructed from concatenated multi-locus sequence typing alleles and clustered with a novel k-medoids approach. One hundred isolates representing 22 Bcc species, plus B. gladioli, were assessed for bacterial identifications using the two MALDI-TOF systems.Results. At the genus level, 100 and 97.0 % of isolates were confidently identified as Burkholderia by the Biotyper and VITEK MS systems, respectively; moreover, 26.0 and 67.0 % of the isolates were correctly identified to the species level, respectively. In many, but not all, cases of species misidentification or failed identification, a representative library for that species was lacking.Conclusion. Currently available MALDI-TOF systems frequently do not accurately identify Bcc bacteria to the species level.


Assuntos
Burkholderia cepacia/isolamento & purificação , Burkholderia gladioli/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Técnicas de Tipagem Bacteriana/métodos , Burkholderia cepacia/classificação , Burkholderia gladioli/classificação , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Análise de Fourier , Humanos , Tipagem de Sequências Multilocus , Filogenia , Recombinases Rec A/genética , Alinhamento de Sequência
8.
PLoS One ; 15(4): e0231393, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352974

RESUMO

Whole genome sequencing (WGS) was performed on 201 Listeria monocytogenes isolates recovered from 102 of 27,389 refrigerated ready-to-eat (RTE) food samples purchased at retail in U.S. FoodNet sites as part of the 2010-2013 interagency L. monocytogenes Market Basket Survey (Lm MBS). Core genome multi-locus sequence typing (cgMLST) and in-silico analyses were conducted, and these data were analyzed with metadata for isolates from five food groups: produce, seafood, dairy, meat, and combination foods. Six of 201 isolates, from 3 samples, were subsequently confirmed as L. welshimeri. Three samples contained one isolate per sample; mmong the 96 samples that contained two isolates per sample, 3 samples each contained two different strains and 93 samples each contained duplicate isolates. After 93 duplicate isolates were removed, the remaining 102 isolates were delineated into 29 clonal complexes (CCs) or singletons based on their sequence type. The five most prevalent CCs were CC155, CC1, CC5, CC87, and CC321. The Shannon's diversity index for clones per food group ranged from 1.49 for dairy to 2.32 for produce isolates, which were not significantly different in pairwise comparisons. The most common molecular serogroup as determined by in-silico analysis was IIa (45.6%), followed by IIb (27.2%), IVb (20.4%), and IIc (4.9%). The proportions of isolates within lineages I, II, and III were 48.0%, 50.0% and 2.0%, respectively. Full-length inlA was present in 89.3% of isolates. Listeria pathogenicity island 3 (LIPI-3) and LIPI-4 were found in 51% and 30.6% of lineage I isolates, respectively. Stress survival islet 1 (SSI-1) was present in 34.7% of lineage I isolates, 80.4% of lineage II isolates and the 2 lineage III isolates; SSI-2 was present only in the CC121 isolate. Plasmids were found in 48% of isolates, including 24.5% of lineage I isolates and 72.5% of lineage II isolates. Among the plasmid-carrying isolates, 100% contained at least one cadmium resistance cassette and 89.8% contained bcrABC, involved in quaternary ammonium compound tolerance. Multiple clusters of isolates from different food samples were identified by cgMLST which, along with available metadata, could aid in the investigation of possible cross-contamination and persistence events.


Assuntos
Microbiologia de Alimentos , Variação Genética , Listeria monocytogenes/genética , Virulência/genética , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Humanos , Listeria monocytogenes/classificação , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/patogenicidade , Listeriose/patologia , Listeriose/transmissão , Tipagem de Sequências Multilocus , Filogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Sorogrupo , Sequenciamento Completo do Genoma
9.
PLoS One ; 15(5): e0228479, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413030

RESUMO

Terminator is a DNA sequence that gives the RNA polymerase the transcriptional termination signal. Identifying terminators correctly can optimize the genome annotation, more importantly, it has considerable application value in disease diagnosis and therapies. However, accurate prediction methods are deficient and in urgent need. Therefore, we proposed a prediction method "iterb-PPse" for terminators by incorporating 47 nucleotide properties into PseKNC-Ⅰ and PseKNC-Ⅱ and utilizing Extreme Gradient Boosting to predict terminators based on Escherichia coli and Bacillus subtilis. Combing with the preceding methods, we employed three new feature extraction methods K-pwm, Base-content, Nucleotidepro to formulate raw samples. The two-step method was applied to select features. When identifying terminators based on optimized features, we compared five single models as well as 16 ensemble models. As a result, the accuracy of our method on benchmark dataset achieved 99.88%, higher than the existing state-of-the-art predictor iTerm-PseKNC in 100 times five-fold cross-validation test. Its prediction accuracy for two independent datasets reached 94.24% and 99.45% respectively. For the convenience of users, we developed a software on the basis of "iterb-PPse" with the same name. The open software and source code of "iterb-PPse" are available at https://github.com/Sarahyouzi/iterb-PPse.


Assuntos
Análise de Sequência de DNA/métodos , Software , Regiões Terminadoras Genéticas , Bacillus subtilis , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator Rho/metabolismo , Terminação da Transcrição Genética
10.
Nucleic Acids Res ; 48(12): 6403-6412, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32424410

RESUMO

Promoter design remains one of the most important considerations in metabolic engineering and synthetic biology applications. Theoretically, there are 450 possible sequences for a 50-nt promoter, of which naturally occurring promoters make up only a small subset. To explore the vast number of potential sequences, we report a novel AI-based framework for de novo promoter design in Escherichia coli. The model, which was guided by sequence features learned from natural promoters, could capture interactions between nucleotides at different positions and design novel synthetic promoters in silico. We combined a deep generative model that guides the search for artificial sequences with a predictive model to preselect the most promising promoters. The AI-designed promoters were optimized based on the promoter activity in E. coli and the predictive model. After two rounds of optimization, up to 70.8% of the AI-designed promoters were experimentally demonstrated to be functional, and few of them shared significant sequence similarity with the E. coli genome. Our work provided an end-to-end approach to the de novo design of novel promoter elements, indicating the potential to apply deep learning methods to de novo genetic element design.


Assuntos
Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Software , DNA Bacteriano/química , DNA Bacteriano/genética , Aprendizado Profundo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
11.
Food Chem ; 324: 126859, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32344343

RESUMO

Here, we constructed a fast, universal, "turn-on" biosensor for the highly sensitive visual detection of Salmonella based on luminescent DNAzyme and a universal blocking linker Super Polymerase Chain Reaction (S-PCR). The primer in this biosensor was specially designed. The G-quadruplex sequence is attached to the 5' end of the primer by a blocking linker and is blocked in the stem part. The S-PCR amplification releases the G-quadruplex, which is incubated with hemin to form DNAzyme to exert peroxide-like activity. The visible colored products are generated by the addition of 3,3',5,5'-Tetramethylbenzidine. Detection sensitivity is high and, even when the concentration of the Salmonella genome in a sample is as low as 1.5 copies/µL, this color change can be seen with the naked eyes. Moreover, this method is simple and fast, as the S-PCR can be completed in less than 10 min using the newly built equipment and without the need for large instruments.


Assuntos
Técnicas Biossensoriais/métodos , DNA Catalítico/química , Salmonella/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Quadruplex G , Limite de Detecção , Reação em Cadeia da Polimerase , Salmonella/genética
12.
Biochim Biophys Acta Gene Regul Mech ; 1863(5): 194515, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32113983

RESUMO

Positive supercoiling buildup (PSB) is a pervasive phenomenon in the transcriptional programs of Escherichia coli. After finding a range of Gyrase concentrations where the inverse of the transcription rate of a chromosome-integrated gene changes linearly with the inverse of Gyrase concentration, we apply a LineWeaver-Burk plot to dissect the expected in vivo transcription rate in absence of PSB. We validate the estimation by time-lapse microscopy of single-RNA production kinetics of the same gene when single-copy plasmid-borne, shown to be impervious to Gyrase inhibition. Next, we estimate the fraction of time in locked states and number of transcription events prior to locking, which we validate by measurements under Gyrase inhibition. Replacing the gene of interest by one with slower transcription rate decreases the fraction of time in locked states due to PSB. Finally, we combine data from both constructs to infer a range of possible transcription initiation locking kinetics in a chromosomal location, obtainable by tuning the transcription rate. We validate with measurements of transcription activity at different induction levels. This strategy for dissecting transcription initiation locking kinetics due to PSB can contribute to resolve the transcriptional programs of E. coli and in the engineering of synthetic genetic circuits.


Assuntos
Simulação por Computador , DNA Girase/metabolismo , DNA Bacteriano/genética , DNA Super-Helicoidal/genética , Proteínas de Escherichia coli/metabolismo , Iniciação da Transcrição Genética , DNA Bacteriano/química , DNA Super-Helicoidal/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Cinética , Novobiocina/farmacologia , RNA/genética , RNA/metabolismo , Inibidores da Topoisomerase II/farmacologia
13.
PLoS One ; 15(2): e0228591, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023304

RESUMO

Biofilms are currently considered as a predominant lifestyle of many bacteria in nature. While they promote survival of microbes, biofilms also potentially increase the threats to animal and public health in case of pathogenic species. They not only facilitate bacteria transmission and persistence, but also promote spreading of antibiotic resistance leading to chronic infections. In the case of Francisella tularensis, the causative agent of tularemia, biofilms have remained largely enigmatic. Here, applying live and static confocal microscopy, we report growth and ultrastructural organization of the biofilms formed in vitro by these microorganisms over the early transition from coccobacillary into coccoid shape during biofilm assembly. Using selective dispersing agents, we provided evidence for extracellular DNA (eDNA) being a major and conserved structural component of mature biofilms formed by both F. subsp. novicida and a human clinical isolate of F. philomiragia. We also observed a higher physical robustness of F. novicida biofilm as compared to F. philomiragia one, a feature likely promoted by specific polysaccharides. Further, F. novicida biofilms resisted significantly better to ciprofloxacin than their planktonic counterparts. Importantly, when grown in biofilms, both Francisella species survived longer in cold water as compared to free-living bacteria, a trait possibly associated with a gain in fitness in the natural aquatic environment. Overall, this study provides information on survival of Francisella when embedded with biofilms that should improve both the future management of biofilm-related infections and the design of effective strategies to tackle down the problematic issue of bacteria persistence in aquatic ecosystems.


Assuntos
Biofilmes , Farmacorresistência Bacteriana , Francisella/fisiologia , Água Doce/microbiologia , Adaptação Fisiológica , Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , Sequência Conservada , DNA Bacteriano/química , Francisella/efeitos dos fármacos , Francisella/genética , Francisella/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos
14.
PLoS One ; 15(2): e0229740, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32106263

RESUMO

Bacterial production has been often estimated from DNA synthesis rates by using tritium-labeled thymidine. Some bacteria species cannot incorporate extracellular thymidine into their DNA, suggesting their biomass production might be overlooked when using the conventional method. In the present study, to evaluate appropriateness of deoxyribonucleosides for evaluating bacterial production of natural bacterial communities from the viewpoint of DNA synthesis, incorporation rates of four deoxyribonucleosides (thymidine, deoxyadenosine, deoxyguanosine and deoxycytidine) labeled by nitrogen stable isotope (15N) into bacterial DNA were examined in both ocean (Sagami Bay) and freshwater (Lake Kasumigaura) ecosystems in July 2015 and January 2016. In most stations in Sagami Bay and Lake Kasumigaura, we found that incorporation rates of deoxyguanosine were the highest among those of the four deoxyribonucleosides, and the incorporation rate of deoxyguanosine was approximately 2.5 times higher than that of thymidine. Whereas, incorporation rates of deoxyadenosine and deoxycytidine were 0.9 and 0.2 times higher than that of thymidine. These results clearly suggest that the numbers of bacterial species which can incorporate exogenous deoxyguanosine into their DNA are relatively greater as compared to the other deoxyribonucleosides, and measurement of bacterial production using deoxyguanosine more likely reflects larger numbers of bacterial species productions.


Assuntos
DNA Bacteriano/biossíntese , DNA Bacteriano/química , Isótopos de Nitrogênio/metabolismo , Baías/microbiologia , Biomassa , Desoxiadenosinas/metabolismo , Desoxicitidina/metabolismo , Desoxiguanosina/metabolismo , Ecossistema , Japão , Cinética , Lagos/microbiologia , Consórcios Microbianos/fisiologia , Timidina/metabolismo
15.
Cell ; 180(4): 703-716.e18, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32059782

RESUMO

The three-dimensional structures of chromosomes are increasingly being recognized as playing a major role in cellular regulatory states. The efficiency and promiscuity of phage Mu transposition was exploited to directly measure in vivo interactions between genomic loci in E. coli. Two global organizing principles have emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation.


Assuntos
Bacteriófago mu/genética , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/química , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Conformação de Ácido Nucleico , Transposases/genética , Transposases/metabolismo
16.
Epidemiol Infect ; 148: e49, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32054545

RESUMO

A new fast-growing mycobacterium, designated strain QGD101T, was isolated from the sputum of an 84-year-old man suspected of tuberculosis in Wuhan Medical Treatment Center, Hubei, China. This strain was a gram-staining-negative, aerobic, non-spore-forming and catalase-positive bacterium, which was further identified as the NTM by PNB and TCH tests. The moxifloxacin and levofloxacin exhibited strong suppressing function against QGD101T with MIC values of 0.06 and 0.125 µg/ml after drug susceptibility testing of six main antimicrobial agents on mycobacteria. Based on the sequence analysis of 16S rRNA, rpoB, hsp65 and 16S-23S rRNA internal transcribed spacer, the strain QGD101T could not be identified to a species level. Mycobacterium moriokaense ATCC43059T that shared the highest 16S rRNA gene sequence similarity (98%) with strain QGD101T was actually different in genomes average nucleotide identity (78.74%). In addition, the major cellular fatty acids of QGD101T were determined as C18:1ω9c, C16:0 and C18:2ω6c. The DNA G + C content was 64.9% measured by high performance liquid chromatography. Therefore, the phenotypic and genotypic characterisation of this strain led us to the conclusion that it represents a novel species of mycobacteria, for which the name Mycobacterium hubeiense sp. nov. (type strain QGD101T = CCTCCAA 2017003T = KCTC39927T) was proposed. Thus, the results of this study are very significant for the clinical diagnosis of tuberculosis and future personalised medicine.


Assuntos
Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Escarro/microbiologia , Idoso de 80 Anos ou mais , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Composição de Bases , Chaperonina 60/genética , China , Análise por Conglomerados , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , RNA Polimerases Dirigidas por DNA/genética , Ácidos Graxos/análise , Humanos , Levofloxacino/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Mycobacterium/genética , Mycobacterium/fisiologia , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
17.
Nat Struct Mol Biol ; 27(1): 71-77, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31907455

RESUMO

The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We report cryo-EM structures of the Escherichia coli RecBCD complex bound to several different DNA forks containing a Chi sequence, including one in which Chi is recognized and others in which it is not. The Chi-recognized structure shows conformational changes in regions of the protein that contact Chi and reveals a tortuous path taken by the DNA. Sequence specificity arises from interactions with both the RecC subunit and the sequence itself. These structures provide molecular details for how Chi is recognized and insights into the changes that occur in response to Chi binding that switch RecBCD from bacteriophage destruction and CRISPR spacer acquisition to constructive host DNA repair.


Assuntos
Reparo do DNA , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Exodesoxirribonuclease V/metabolismo , Bacteriófago lambda/fisiologia , Sequência de Bases , Sítios de Ligação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/ultraestrutura , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Exodesoxirribonuclease V/química , Exodesoxirribonuclease V/ultraestrutura , Simulação de Acoplamento Molecular , Conformação Proteica
18.
Nucleic Acids Res ; 48(4): 2035-2049, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31950157

RESUMO

Negative supercoiling by DNA gyrase is essential for maintaining chromosomal compaction, transcriptional programming, and genetic integrity in bacteria. Questions remain as to how gyrases from different species have evolved profound differences in their kinetics, efficiency, and extent of negative supercoiling. To explore this issue, we analyzed homology-directed mutations in the C-terminal, DNA-wrapping domain of the GyrA subunit of Escherichia coli gyrase (the 'CTD'). The addition or removal of select, conserved basic residues markedly impacts both nucleotide-dependent DNA wrapping and supercoiling by the enzyme. Weakening CTD-DNA interactions slows supercoiling, impairs DNA-dependent ATP hydrolysis, and limits the extent of DNA supercoiling, while simultaneously enhancing decatenation and supercoil relaxation. Conversely, strengthening DNA wrapping does not result in a more extensively supercoiled DNA product, but partially uncouples ATP turnover from strand passage, manifesting in futile cycling. Our findings indicate that the catalytic cycle of E. coli gyrase operates at high thermodynamic efficiency, and that the stability of DNA wrapping by the CTD provides one limit to DNA supercoil introduction, beyond which strand passage competes with ATP-dependent supercoil relaxation. These results highlight a means by which gyrase can evolve distinct homeostatic supercoiling setpoints in a species-specific manner.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Girase/genética , DNA Bacteriano/genética , DNA Super-Helicoidal/química , Trifosfato de Adenosina/química , Catálise , Cromossomos Bacterianos/genética , DNA Girase/química , DNA Bacteriano/química , DNA Super-Helicoidal/genética , Escherichia coli/enzimologia , Modelos Moleculares , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos/genética
19.
Mol Phylogenet Evol ; 145: 106730, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31904510

RESUMO

In a moss samples collected on Madagascar two populations of Paramacrobiotus experimentalis sp. nov. were found. Paramacrobiotus experimentalis sp. nov. with the presence of a microplacoid and areolatus type of eggs is similar to Pam. danielae, Pam. garynahi, Pam. hapukuensis, Pam. peteri, Pam. rioplatensis and Pam. savai, but it differs from them by some morphological and morphometric characters of the eggs. The p-distance between two COI haplotypes of Pam. experimentalis sp. nov. was 0.17%. In turn, the ranges of uncorrected genetic p-distances of all Paramacrobiotus species available in GenBank was from 18.27% (for Pam. lachowskae) to 25.26% (for Pam. arduus) with an average distance of 20.67%. We also found that Pam. experimentalis sp. nov. is bisexual. This observation was congruent on three levels: (i) morphological - specimen size dimorphism; (ii) structural (primary sexual characteristics) - females have an unpaired ovary while males have an unpaired testis and (iii) molecular - heterozygous and homozygous strains of the ITS-2 marker. Although symbiotic associations of hosts with bacteria (including endosymbiotic bacteria) are common in nature and these interactions exert various effects on the evolution, biology and reproductive ecology of hosts, there is still very little information on the bacterial community associated with tardigrades. To fill this gap and characterise the bacterial community of Pam. experimentalis sp. nov. populations and microbiome of its microhabitat, high throughput sequencing of the V3-V4 hypervariable regions in the bacterial 16S rRNA gene fragment was performed. The obtained 16S rRNA gene sequences ranged from 92,665 to 131,163. In total, 135 operational taxonomic units (OTUs) were identified across the rarefied dataset. Overall, both Pam. experimentalis sp. nov. populations were dominated by OTUs ascribed to the phylum Proteobacteria (89-92%) and Firmicutes (6-7%). In the case of samples from tardigrades' laboratory habitat, the most abundant bacterial phylum was Proteobacteria (51-90%) and Bacteroides (9-48%). In all compared microbiome profiles, only 16 of 137 OTUs were shared. We found also significant differences in beta diversity between the partly species-specific microbiome of Pam. experimentalis sp. nov. and its culturing environment. Two OTUs belonging to a putative bacterial endosymbiont were identified - Rickettsiales and Polynucleobacter. We also demonstrated that each bacterial community was rich in genes involved in membrane transport, amino acid metabolism, and carbohydrate metabolism.


Assuntos
Microbiota , Tardígrados/classificação , Animais , Bacteroides/genética , Bacteroides/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/classificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Madagáscar , Masculino , Mitocôndrias/genética , Filogenia , Proteobactérias/genética , Proteobactérias/isolamento & purificação , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 18S/classificação , RNA Ribossômico 18S/genética , Simbiose , Tardígrados/genética , Tardígrados/microbiologia
20.
Epidemiol Infect ; 148: e6, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31933451

RESUMO

Cervids represent a mammal group which plays an important role in the maintenance of ecological balance. Recent studies have highlighted the role of these species as reservoirs for several arthropods-borne pathogens. Globally, hemotropic mycoplasmas (haemoplasmas) are emerging or remerging bacteria that attach to red blood cells of several mammals species causing hemolytic anaemia. Therefore, the aim of this study was to investigate the occurrence and assess the phylogenetic positioning of Mycoplasma ovis in free-ranging deer from Brazil. Using a polymerase chain reaction targeting the 16S rRNA region, 18 (40%) out of 45 sampled deer were positive to M. ovis. Among the nine sequences analysed, four distinct genotypes were identified. The sequences detected in the present study were closely related to sequences previously identified in deer from Brazil and the USA. On the other hand, the Neighbour-Net network analysis showed that the human-associated M. ovis genotypes were related to genotypes detected in sheep and goats. The present study shows, for the first time, the occurrence of M. ovis in Mazama gouazoubira and Mazama bororo deer species, expanding the knowledge on the hosts harbouring this haemoplasma species. Once several deer species have your population in decline, additional studies are needed to evaluate the pathogenicity of M. ovis among deer populations around the world and assess its potential as reservoir hosts to human infections.


Assuntos
Doenças dos Animais/epidemiologia , Doenças dos Animais/microbiologia , Cervos/microbiologia , Variação Genética , Infecções por Mycoplasma/veterinária , Mycoplasma/classificação , Mycoplasma/isolamento & purificação , Animais , Brasil , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genótipo , Mycoplasma/genética , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
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